Daptomycin formulations and uses thereof

ABSTRACT

Lyophilized daptomycin formulations having improved reconstitution times are provided. The lyophilized daptomycin formulations include an additive, which can be a pharmaceutically acceptable antioxidant, a pharmaceutically acceptable organic acid or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable glucose derivative or a pharmaceutically acceptable salt thereof, or a combination thereof. Also provided are methods of methods of preparing the lyophilized daptomycin formulations, and methods of treating bacterial infections and treating or preventing biofilms by using the lyophilized daptomycin formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Patent Application under 35U.S.C. §371 of International Application No. PCT/IB2013/002191, filed onSep. 11, 2013, which claims priority under 35 U.S.C. §119 to U.S.Application Ser. No. 61/699,570, filed Sep. 11, 2012, and 61/839,699,filed Jun. 26, 2013, the disclosures of which are hereby incorporated byreference in their entireties, and to each of which priority is claimed.

FIELD

The presently disclosed subject matter relates to lyophilized daptomycinformulations having improved reconstitution times and methods ofpreparing thereof. The presently disclosed subject matter also relatesto methods of treating a bacterial infection in a subject by using thelyophilized daptomycin formulations.

BACKGROUND

Daptomycin (I) is a cyclic lipopeptide derived from a natural product ofStreptomyces roseosporus. The daptomycin comprises an asparagine (Asn)residue in the D configuration. Daptomycin has been used for treatingcomplicated skin and skin structure infections (cSSSI) caused bysusceptible isolates of the following gram-positive bacteria:Staphylococcus aureus (including methicillin-resistant isolates),Streptococcus pyogenes, Streptococcus agalactiae, Streptococcusdysgalactiae subsp. equisimilis, and Enterococcus faecalis(vancomycin-susceptible isolates only).

Daptomycin has also been used for treating Staphylococcus aureusbloodstream infections (bacteremia), including those with right-sidedinfective endocarditis, caused by methicillin-susceptible andmethicillin-resistant isolates. Daptomycin's bactericidal effects stemfrom its ability to rapidly depolarize the membrane potential ofgram-positive bacteria, which causes inhibition of DNA, RNA and proteinsynthesis, and results in cell death. The bactericidal effect ofdaptomycin is rapid, with greater than 99.9% of both MRSA and MSSAbacteria dead in less than one hour.

Daptomycin has also been used for biofilm treatment includingcatheter-related bloodstream infections (CRBSI) due to gram-positivebacteria. Particularly, daptomycin may be used for central venouscatheter salvage for S. aureus and S. epidermidis infected catheters.

Daptomycin is commercially available as CUBICIN® (“the CUBICIN®product”, Cubist Pharmaceuticals, Inc., Lexington, Mass.) and issupplied as a sterile, lyophilized powder. CUBICIN® is reconstituted insodium chloride for parenteral injection. Stability studies have shownthat the reconstituted solution is stable in a vial for 12 hours at roomtemperature and up to 48 hours if stored under refrigeration at 2 to 8°C. However, after this time, or at higher temperatures, daptomycinbegins to degrade.

A major shortcoming of the commercially available daptomycin is that thereconstitution time of the CUBICIN® product is long and is typically inthe range of about 15 to 45 minutes depending on the reconstitutionprocedure. This reconstitution time is not ideal in a therapeuticsetting with respect to ease and efficiency of administration. Such longreconstitution time also increases the risk of inadvertent incompletedissolution prior to administration and additionally increases thelikelihood that the daptomycin will degrade prior to patientadministration.

There are a number of daptomycin degradation products that have beenidentified. The major degradants of daptomycin are anhydro-daptomycinderivatives in which an α-aspartyl group is transpeptidated to ananhydrosuccinamido group, β-isomer of daptomycin in which the compoundcontains a β-aspartyl group instead of an α-aspartyl group and thelactone hydrolysis product of daptomycin in which one of the estersmoieties is hydrolysed. The degradation pathway of daptomycin isdescribed in U.S. Patent Publication No. 2007/191280.

High performance liquid chromatography (HPLC) of the reconstitutedlyophilized powder can be used to determine the level of daptomycinrelative to daptomycin degradants. Such comparison can thereby providean indication of the stability of the daptomycin in the formulation. Forexample, International Patent Publication No. WO2011/063419 disclosessolid daptomycin preparations with improved reconstitution times andstability profiles relative to the CUBICIN® product. This is achievedwhen sugars, such as sucrose, and a phosphate buffer are introduced intothe formulation. The formulations have a pH of about 6.5 to about 7.5.The sugars used can be non-reducing sugars and are included in theformulations in an amount of about 2.5% w/v to about 25% w/v. Forexample, the sugars are included in amounts of 15% w/v and 20% w/v.

There is still a need for alternative solid lyophilized daptomycinformulations that exhibit rapid reconstitution times, preferably in lessthan about 5 minutes, in a pharmaceutically accepted diluent.Additionally, there still remains need for solid daptomycin formulationsthat exhibit rapid reconstitution times and have improved stability inboth solid and reconstituted forms. Such a solid formulation would beadvantageous, as it would provide for a longer shelf life, a lessenedrequirement for refrigerated storage and a reduced handling time inreconstitution of the product before use. Such a formulation would alsoprovide for more rapid administration and a more reliable dosing ofdaptomycin due to the presence of fewer impurities resulting fromdaptomycin degradation.

SUMMARY

The presently disclosed subject matter provides lyophilized daptomycinformulations having improved reconstitution times and methods ofpreparing thereof. Also provided are methods of treating a bacterialinfection in a subject by using the lyophilized daptomycin formulations.The presently disclosed subject matter further provides methods oftreating or preventing a biofilm by using the lyophilized daptomycinformulations.

The presently disclosed subject matter provides a lyophilized daptomycinformulation comprising an additive selected from the group consisting ofpharmaceutically acceptable antioxidants, pharmaceutically acceptableorganic acids and pharmaceutically acceptable salts thereof,pharmaceutically acceptable glucose derivatives and pharmaceuticallyacceptable salts thereof, and combinations thereof. In certainembodiments, the lyophilized daptomycin formulation includes from about200 mg to about 600 mg of daptomycin. In certain embodiments, thelyophilized daptomycin formulation includes from about 0.01 mM to about500 mM of the additive.

In one embodiment, the pharmaceutically acceptable antioxidant isascorbic acid. The pharmaceutically acceptable organic acid can beselected from the group consisting of monocarboxylic organic acids,dicarboxylic organic acids, hydroxyl substituted dicarboxylic organicacids, tricarboxylic organic acids, hydroxyl substituted tricarboxylicorganic acids, tetracarboxylic organic acids, and combinations thereof.In certain embodiments, the pharmaceutically acceptable organic acid isa hydroxyl substituted tricarboxylic organic acid. In one embodiment,the hydroxyl substituted tricarboxylic organic acid is citric acid. Inone embodiment, the pharmaceutically acceptable glucose derivative isacetyl glucosamine.

The lyophilized daptomycin formulation can be reconstituted in apharmaceutically acceptable diluent. In one embodiment, the lyophilizeddaptomycin formulation is reconstituted in a pharmaceutically acceptablediluent in less than about 5 minutes. The concentration of daptomycin inthe reconstituted lyophilized daptomycin formulation can be from about20 mg/mL to about 100 mg/mL.

The concentration of the additive in the reconstituted lyophilizeddaptomycin formulation can be from about 1 mM to about 500 mM. In oneembodiment, the concentration of the additive in the reconstitutedlyophilized daptomycin formulation is about 237.5 mM. In one embodiment,the concentration of the additive in the reconstituted lyophilizeddaptomycin formulation is about 300 mM. In certain embodiments, theconcentration of the additive in the reconstituted lyophilizeddaptomycin formulation is from about 1 mg/mL to about 500 mg/mL.

The pH of the reconstituted lyophilized daptomycin formulation can befrom about 4.0 to about 5.0. In one embodiment, the pH of thereconstituted lyophilized daptomycin formulation is about 4.7.

The diluent can be selected from the group consisting of sterile waterfor injection, bacteriostatic water for injection, 0.45% sodium chloridesolution for injection, 0.9% sodium chloride solution for injection,Ringer's solution, lactated Ringer's solution, and combinations thereof.In one embodiment, the diluent is 0.9% sterile sodium chloride solutionfor injection. In another embodiment, the diluent is sterile water forinjection.

The presently disclosed subject matter also provides a method oftreating a bacterial infection in a subject. The method includesadministering to a subject in need thereof, an effective amount of theabove-disclosed lyophilized daptomycin formulation. Additionally, thepresently disclosed subject matter provides a method of treating orpreventing a biofilm. The method includes exposing a surface of a deviceto a solution of an effective amount of the above-disclosed lyophilizeddaptomycin formulation.

The presently disclosed subject matter further provides methods forpreparing lyophilized daptomycin formulations. In one embodiment, themethod includes (a) forming an aqueous solution of daptomycin and anadditive, which is selected from the group consisting ofpharmaceutically acceptable organic acids and pharmaceuticallyacceptable salts thereof, pharmaceutically acceptable glucosederivatives and pharmaceutically acceptable salts thereof, andcombinations thereof; (b) adjusting the pH to about 4.0 to about 5.0;and (c) lyophilising the solution to obtain a lyophilisate. In anotherembodiment, the method includes (a) forming an aqueous solution ofdaptomycin at a pH of about 4.0 to about 5.0; (b) dissolving an additiveselected from the group consisting of pharmaceutically acceptableorganic acids and pharmaceutically acceptable salts thereof,pharmaceutically acceptable glucose derivatives and pharmaceuticallyacceptable salts thereof, and combinations thereof in the aqueoussolution of the daptomycin; (c) adjusting the pH to about 4.0 to about5.0; and (d) lyophilising the solution to obtain a powder.

DETAILED DESCRIPTION Definitions

The examples provided in the definitions present in the presentapplication are non-inclusive unless otherwise stated. They include butare not limited to the recited examples.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 3 or more than 3 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value.

As used herein, the term “osmolality” of a solution means the number ofosmoles of solute per kilogram of solvent. Osmolality is a measure ofthe osmotic pressure exerted by a real solution across a semi-permeablemembrane. It can be measured by use of a property of the solution thatis dependent only on the particle concentration. These propertiesinclude vapour pressure depression, freezing point and boiling pointdepression, osmotic pressure collectively referred to as colligativeproperties. The osmolality of a solution is typically determined mostaccurately and conveniently by measuring freezing point depression.

As used herein, the term “lyophilized” means a stabilizing process usedto remove a solvent from tissue, blood, serum, pharmaceuticalformulations, or other biological substances; at low temperaturesthrough a process of sublimation (primary drying) and then desorption(secondary drying). A lyophilized formulation can be reconstituted in asimple manner to give a ready-to-use solution which contains no visibleparticles by addition of a diluent.

Lyophilized Daptomycin Formulations

The presently disclosed subject matter provides a lyophilized daptomycinformulation including an additive. The additive can be apharmaceutically acceptable antioxidant, a pharmaceutically acceptableorganic acid and a pharmaceutically acceptable salt thereof, apharmaceutically acceptable glucose derivative and a pharmaceuticallyacceptable salt thereof, and a combination thereof.

The lyophilized daptomycin formulation of the presently disclosedsubject matter displays rapid reconstitution times in a pharmaceuticallyacceptable diluent. Furthermore, the inclusion of an additive in thedaptomycin formulations does not adversely affect the stability of theformulations with respect to the CUBICIN® product. The lyophilizeddaptomycin formulations of the presently disclosed subject matterdisplay good pharmaceutical elegance and upon reconstitution displayminimal foaming. The presence of foaming upon reconstitution can beproblematic with daptomycin solutions due to its amphiphilic properties,particularly in a clinical setting wherein foaming of the formulationaffects the reconstitution time, the homogeneity of the reconstitutedsolution, as well as the ease and accuracy of delivering the formulationto a patient. The lyophilized daptomycin formulations of the presentlydisclosed subject matter thereby allow for simple and rapidreconstitution and provide daptomycin solution formulations withimproved homogeneity that can be more easily and precisely delivered toa patient.

Additives

The additives suitable for use in the formulations of the presentlydisclosed subject matter can be pharmaceutically acceptableantioxidants, pharmaceutically acceptable organic acids or saltsthereof, pharmaceutically acceptable antioxidants, pharmaceuticallyacceptable glucose derivatives or salts thereof, or combinationsthereof.

The additives suitable for use in the formulations of the presentlydisclosed subject matter can be pharmaceutically acceptableantioxidants. Suitable antioxidants include, but are not limited to,ascorbic acid, monothioglycerol, L-cysteine, thioglycolic acid, sodiummetabisulfite, sodium EDTA, monoethanolamine gentisate, sodiumformaldehyde sulfoxylate and sodium bisulfite. In one embodiment, theantioxidant is ascorbic acid.

The organic acids suitable for use in the presently disclosed subjectmatter include, but are not limited to, monocarboxylic organic acids,dicarboxylic organic acids, hydroxyl substituted dicarboxylic organicacids, tricarboxylic organic acids, and tetracarboxylic organic acids.

In one embodiment, the dicarboxylic organic acid is a hydroxylsubstituted dicarboxylic organic acid. In another embodiment, thetricarboxylic organic acid is a hydroxyl substituted tricarboxylicorganic acid.

Monocarboxylic organic acids suitable for use in the presently disclosedsubject matter include, but are not limited to, acetic acid, lacticacid, thiolactic acid, glycolic acid, butyric acid, isobutyric acid,glyceric acid, oxaloacetic acid, pyruvic acid, propionic acid, valericacid, pivalic acid and benzoic acid. In one embodiment, themonocarboxylic acid is acetic acid. In another embodiment, thepharmaceutically acceptable salt of a monocarboxylic organic acid issodium acetate. In yet another embodiment, the pharmaceuticallyacceptable salt of a monocarboxylic organic acid is potassium acetate.

Dicarboxylic organic acids suitable for use in the presently disclosedsubject matter include, but are not limited to, malonic acid, succinicacid, adipic acid, maleic acid and glutaric acid. In one embodiment, thedicarboxylic organic acid is succinic acid.

Hydroxyl substituted dicarboxylic organic acids suitable for use in thepresently disclosed subject matter include, but are not limited to,glucaric acid, α-hydroxyglutaric acid, gluconic acid, malic acid andtartaric acid. In one embodiment, the hydroxyl substituted dicarboxylicorganic acid is tartaric acid.

Tricarboxylic organic acids suitable for use in the presently disclosedsubject matter include, but are not limited to, aconitic acid andoxalosuccinic acid.

Hydroxyl substituted tricarboxylic organic acids suitable for use in thepresently disclosed subject matter include, but are not limited to,citric acid, isocitric acid, homocitric acid and hydroxycitric acid. Inone embodiment, the hydroxyl substituted tricarboxylic organic acid iscitric acid.

Tetracarboxylic organic acids suitable for use in the presentlydisclosed subject matter include, but are not limited to, edetic acid,ethylene tetracarboxylic acid and ethylene glycol tetraacetic acid. Thesuitable pharmaceutically acceptable salts of tetracarboxylic organicacids include, but are not limited to, disodium edetate and tetrasodiumedetate. In one embodiment, the tetracarboxylic organic acid is edeticacid. In another embodiment, the pharmaceutically acceptable salt of atetracarboxylic organic acid is disodium edetate.

Furthermore, the additives suitable for use in the formulations of thepresently disclosed subject matter can be pharmaceutically acceptableglucose derivatives or salts thereof. In one embodiment, the glucosederivative is acetyl glucosamine.

When the additive is a pharmaceutically acceptable salt of apharmaceutically acceptable organic acid or a pharmaceuticallyacceptable glucose derivative, the salt may be formed in situ by theaddition of a pharmaceutically acceptable base to an acid solution.Alternatively, the salt may be added directly to the formulation. Thecation of the salt includes, but is not limited to, sodium, potassium,lithium, calcium, magnesium, zinc, ammonium, alkylammonium (such astriethylammonium), alkoxyammonium (such as ethanolammonium andethanediaminium), choline and amino acids (such as arginine, lysine orhistidine).

The lyophilized daptomycin formulations of the presently disclosedsubject matter can include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 mM, or a rangeincluding any of two of those integers, of an additive.

In some embodiments, the lyophilized daptomycin formulation includesfrom about 0.01 mM to about 500 mM of an additive. The lyophilizeddaptomycin formulation can include from about 0.05 mM to about 450 mM,from about 0.05 mM to about 300 mM, or from about 0.05 mM to about 100mM, of an additive.

In one embodiment, the additive is a combination of a pharmaceuticallyacceptable antioxidant, a pharmaceutically acceptable organic acid, apharmaceutically acceptable salt thereof, a pharmaceutically acceptableglucose derivative, and/or a pharmaceutically acceptable salt thereof.The concentration of the additive is the sum of the concentrations ofthe antioxidant, the organic acid, the salt thereof, the glucosederivative, and/or the salt thereof expressed in terms of minimolar (mM)or mass per volume (mg/mL). In one embodiment, the salt is the conjugatebase of the organic acid so as to form a buffer solution.

In certain embodiments, the additive is used at a concentration thatdoes not destabilise the daptomycin and preferably aids stability of thedaptomycin. The stability of daptomycin will depend on the intendedshelf life of the pharmaceutical formulation and the manipulation priorto administration. In one embodiment, the daptomycin formulations of thepresently disclosed subject matter are at least as stable as theCUBICIN®) lyophilized powder.

Daptomycin

The CUBICIN® product includes 250, 350 or 500 mg of daptomycin. Thelyophilized daptomycin formulations of the presently disclosed subjectmatter include from about 200 mg to about 600 mg of daptomycin. In someembodiments, the lyophilized daptomycin formulations include from about200 mg to about 300 mg of daptomycin. In one embodiment, the lyophilizeddaptomycin formulations include about 250 mg of daptomycin. In someembodiments, the lyophilized daptomycin formulations include from about300 mg to about 400 mg of daptomycin. In one embodiment, the lyophilizeddaptomycin formulations include about 350 mg of daptomycin. In certainembodiments, the lyophilized daptomycin formulations include from about450 mg to about 550 mg of daptomycin. In one embodiment, the lyophilizeddaptomycin formulations include about 500 mg of daptomycin.

Optional Components

Further optional components can be included in the lyophilizeddaptomycin formulations. Such optional components include, but are notlimited to, buffering agents, stabilisers, solubilisers, crystallisationinhibitors, surfactants and tonicifying agents.

The lyophilized daptomycin formulations of the presently disclosedsubject matter can optionally include one or more buffering agents.Suitable buffering agents include, but are not limited to, phosphatebuffers, sulfonic acids and Tris buffers. Specific buffers includesodium or potassium salts of phosphoric acid (such as disodium hydrogenphosphate), 2-(N-morpholino)ethanesulfonic acid (MES),N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES),N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),3-(N,N-Bis[2-hydroxyethl]amino)-2-hydroxypropanesulfonic acid (DIPSO),2-hydroxy-3-[trishydroxymethyl)methylamine]-1-propanesulfonic acid(TAPSO), N-(2-acetamido)2-aminoethanesulfonic acid (ACES),1,4-piperazinediethanesulfonic acid (PIPES),3-(N-morpholino)propanesulfonic acid (MOPS),β-hydroxy-4-morpholinepropanesulfonic acid (MOPSO),N-(2-acetamido)-iminodiacetic acid (ADA) and2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (BIS-TRIS).The buffering agent can be added in an amount of about 0.01 mM to about500 mM to the lyophilized daptomycin formulations.

The lyophilized daptomycin formulations of the presently disclosedsubject matter can optionally include one or more stabilisers. Suitablestabilising agents include, but are not limited to, sugars (such assucrose, trehalose and dextran), amino acids (such as arginine, glycineand histidine), polyvinylpyrrolidones (povidone) and polyols (such asmannitol and polymeric polyol surfactants, e.g., Pluronic®.

Reconstitution of Lyophilized Daptomycin Formulations

The lyophilized daptomycin formulations of the presently disclosedsubject matter display rapid reconstitution times in a pharmaceuticallyacceptable diluent. In one embodiment, the lyophilized daptomycinformulations are reconstituted in a pharmaceutically acceptable diluentin less than about 5 minutes. In one embodiment, the lyophilizeddaptomycin formulations are reconstituted in a pharmaceuticallyacceptable diluent in less than about 4 minutes. In one embodiment, thelyophilized daptomycin formulations are reconstituted in apharmaceutically acceptable diluent in less than about 3 minutes. Inanother embodiment, the lyophilized daptomycin formulations arereconstituted in a pharmaceutically acceptable diluent in less thanabout 2 minutes. In yet another embodiment, the lyophilized daptomycinformulations are reconstituted in a pharmaceutically acceptable diluentin less than about 1 minute. The lyophilized daptomycin formulations ofthe presently disclosed subject matter have improved reconstitutiontimes compared to the CUBICIN® product.

The CUBICIN® product is reconstituted in 0.9% sterile sodium chloridefor injection. The lyophilized daptomycin formulations of the presentlydisclosed subject matter can be reconstituted with one or morepharmaceutically acceptable diluents to provide a solution suitable foradministration. Pharmaceutically acceptable diluents of the presentlydisclosed subject matter include, but are not limited to, sterile waterfor injection, bacteriostatic water for injection, 0.45% sodium chloridesolution for injection and 0.9% sodium chloride solution for injection,Ringer's solution and lactated Ringer's solution. In one embodiment, thelyophilized daptomycin formulations are reconstituted in 0.9% sterilesodium chloride solution for injection. In another embodiment, thelyophilized daptomycin formulations are reconstituted in sterile waterfor injection.

The lyophilized daptomycin formulations of the presently disclosedsubject matter can be reconstituted by adding the pharmaceuticallyacceptable diluent(s) to the lyophilized daptomycin formulation toprovide the desired concentration for direct administration or furtherdilution for administration by infusion. In some embodiments, the volumeof the pharmaceutically acceptable diluent(s) added to the lyophilizeddaptomycin formulation is from about 5 mL to about 15 mL. In someembodiments, the volume of the pharmaceutically acceptable diluent(s)added to the lyophilized daptomycin formulation is from about 8 mL toabout 12 mL. In one embodiment, the volume of the pharmaceuticallyacceptable diluent(s) added to the lyophilized daptomycin formulation isabout 10 mL.

The lyophilized daptomycin formulations of the presently disclosedsubject matter can be reconstituted by any suitable methods known to oneof ordinary skill in the art. In one embodiment, 10 mL of 0.9% sterilesodium chloride for injection is added slowly to a vial including 500 mgof the lyophilized daptomycin formulation of the presently disclosedsubject matter. The resultant mixture is rotated to ensure all of theformulation is wetted and then allowed to stand undisturbed for about 2minutes. The vial is then gently rotated or swirled intermittently asneeded, to obtain a completely reconstituted solution. Additionally andalternatively, the reconstitution method includes quickly adding adiluent to a vessel including a lyophilized daptomycin formulation ofthe presently disclosed subject matter, followed by swirling of thevessel if required. In some embodiments, the diluent is added in aperiod of about 1-60 seconds. In some embodiments, the diluent is addedin a period of about 1-30 seconds. In one embodiment, the diluent isadded in less than about 20 seconds. In one embodiment, after adding thediluent to the presently disclosed daptomycin formulation, the vesselincluding the daptomycin is swirled for about 1 minute and allowed tostand for about 3-5 minutes until clear.

Upon reconstitution in a pharmaceutically acceptable diluent, theCUBICIN® product includes 50 mg/mL daptomycin. Upon reconstitution in apharmaceutically acceptable diluent, when provided in a vial, thedaptomycin formulations of the presently disclosed subject matterinclude daptomycin at a concentration of from about 20 mg/mL to about100 mg/mL, e.g., from about 20 mg/mL to about 30 mg/mL, from about 30mg/mL to about 40 mg/mL, from about 40 mg/mL to about 50 mg/mL, fromabout 50 mg/mL to about 60 mg/mL, from about 60 mg/mL to about 70 mg/mL,from about 70 mg/mL to about 80 mg/mL, from about 80 mg/mL to about 90mg/mL, or from about 90 mg/mL to about 100 mg/mL. In one embodiment,upon reconstitution in a pharmaceutically acceptable diluent, whenprovided in a vial, the daptomycin formulations include daptomycin at aconcentration of about 50 mg/mL. In another embodiment, uponreconstitution in a pharmaceutically acceptable diluent, when providedin a vial, the daptomycin formulations include daptomycin at aconcentration of about 62.5 mg/mL.

The reconstituted daptomycin formulation can be further diluted in apharmaceutically acceptable diluent for administration to a subject.Pharmaceutically acceptable diluents include, but are not limited to,sterile water for injection, bacteriostatic water for injection, 0.45%sodium chloride solution for injection, 0.9% sodium chloride solutionfor injection, Ringer's solution and lactated Ringer's solution. In oneembodiment, when the reconstituted daptomycin formulation is furtherdiluted for administration to a subject, the final daptomycinconcentration is from about 2.5 to about 20 mg/mL.

Upon reconstitution in a pharmaceutically acceptable diluent, thedaptomycin formulations of the presently disclosed subject matterinclude an additive at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475 and 500 mM, or a range including anyof two of those integers. In some embodiments, upon reconstitution in apharmaceutically acceptable diluent, the presently disclosed daptomycinformulations include an additive at a concentration of from about 1 mMto about 500 mM, from 1 mM to about 250 mM, or from about 1 mM to about150 mM. For example, the concentration of the additive in thereconstituted daptomycin formulations can be from 1 mM to about 50 mM,from about 50 mM to about 100 mM (e.g., from about 80 mM to about 120mM), from about 100 mM to about 150 mM, from about 150 mM to about 200mM, from about 200 mM to about 250 mM (e.g., from about 200 mM to about240 mM, from about 220 mM to about 240 mM, from about 230 mM to about240 mM, or from about 240 mM to about 250 mM), from about 250 mM toabout 350 mM (e.g., from about 250 mM to about 340 mM, from about 260 mMto about 330 mM, from about 270 mM to about 320 mM, from about 280 mM toabout 310 mM, from about 290 mM to about 300 mM, or from about 340 mM toabout 350 mM), or from about 350 mM to about 400 mM, from about 400 mMto about 450 mM, or from about 450 mM to about 500 mM. In certainembodiments, the concentration of the additive in the reconstitutedlyophilized daptomycin formulation is about 237.5 mM. In certainembodiments, the concentration of the additive in the reconstitutedlyophilized daptomycin formulation is about 300 mM. In some embodiments,the concentration of the additive in the reconstituted lyophilizeddaptomycin formulation is about 500 mM.

In one embodiment, the additive is ascorbic acid. The concentration ofascorbic acid in the reconstituted lyophilized daptomycin formulation isabout 237.5 mM. Additionally or alternatively, the concentration ofascorbic acid in the reconstituted lyophilized daptomycin formulation isabout 300 mM.

In one embodiment, the additive is succinic acid. The concentration ofsuccinic acid in the reconstituted formulation is from about 1 mM toabout 500 mM, from about 1 mM to about 250 mM, or from about 80 mM toabout 120 mM.

In one embodiment, the additive is tartaric acid. The concentration oftartaric acid in the reconstituted formulation is from about 1 mM toabout 500 mM, from about 1 mM to about 250 mM, or from about 80 mM toabout 120 mM.

In one embodiment, the additive is citric acid. The concentration ofcitric acid in the reconstituted formulation is from about 1 mM to about500 mM, from about 1 mM to about 250 mM, or from about 25 mM to about 75mM. In one embodiment, the concentration of citric acid in thereconstituted formulation is about 237.5 mM. Additionally oralternatively, the concentration of citric acid in the reconstitutedformulation is about 300 mM. The concentration of citric acid in thereconstituted formulation can also be about 500 mM.

In one embodiment, the additive is edetic acid. The concentration ofedetic acid in the reconstituted formulation is from about 1 to about500 mM, from about 1 to about 250 mM, or from about 50 mM to about 100mM.

In another embodiment, the additive is acetyl glucosamine. Theconcentration of acetyl glucosamine in the reconstituted lyophilizeddaptomycin formulation is about 237.5 mM. Additionally or alternatively,the concentration of acetyl glucosamine in the reconstituted lyophilizeddaptomycin formulation is about 300 mM.

In certain embodiments, upon reconstitution in a pharmaceuticallyacceptable diluent, the presently disclosed daptomycin formulationsinclude an additive at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475 and 500 mg/mL, or a range includingany of two of those integers. In one embodiment, upon reconstitution ina pharmaceutically acceptable diluent, the presently discloseddaptomycin formulations include an additive at a concentration of fromabout 1 mg/mL to about 500 mg/mL, from about 1 mg/mL to about 250 mg/mL,or from about 1 mg/mL to about 100 mg/mL. For example, the concentrationof the additive in the reconstituted daptomycin formulations can be fromabout 1 mg/mL to about 50 mg/mL (e.g., from about 10 mg/mL to about 50mg/mL, from about 20 mg/mL to about 50 mg/mL, from about 30 mg/mL toabout 50 mg/mL, or from about 40 mg/mL to about 50 mg/mL), from about 50mg/mL to about 100 mg/mL (e.g., from about 50 mg/mL to about 90 mg/mL,from about 50 mg/mL to about 80 mg/mL, from about 50 mg/mL to about 70mg/mL, from about 50 mg/mL to about 60 mg/mL, from about 60 mg/mL toabout 70 mg/mL, or from about 90 mg/mL to about 100 mg/mL), from about100 mg/mL to about 150 mg/mL, from about 150 mg/mL to about 200 mg/mL,from about 200 mg/mL to about 250 mg/mL, from about 250 mg/mL to about300 mg/mL, from about 300 mg/mL to about 350 mg/mL, from about 350 mg/mLto about 400 mg/mL, from about 400 mg/mL to about 450 mg/mL, or from 450mg/mL to about 500 mg/mL.

In one embodiment, the additive is ascorbic acid. The concentration ofascorbic acid in the reconstituted lyophilized daptomycin formulation isabout 41.8 mg/mL. Additionally or alternatively, the concentration ofascorbic acid in the reconstituted lyophilized daptomycin formulation isabout 52.8 mg/mL.

In one embodiment, the additive is succinic acid. The concentration ofsuccinic acid in the reconstituted formulation is from about 1 mg/mL toabout 500 mg/mL, from about 1 mg/mL to about 250 mg/mL, or from about 1mg/mL to about 50 mg/mL.

In one embodiment, the additive is tartaric acid. The concentration oftartaric acid in the reconstituted formulation is from about 1 mg/mL toabout 500 mg/mL, from about 1 mg/mL to about 250 mg/mL, or from about 1mg/mL to about 50 mg/mL.

In one embodiment, the additive is citric acid. The concentration ofcitric acid in the reconstituted formulation can be from about 1 mg/mLto about 500 mg/mL, from about 1 mg/mL to about 250 mg/mL, or from about1 mg/mL to about 50 mg/mL. In one embodiment, the concentration ofcitric acid in the reconstituted lyophilized daptomycin formulation isabout 45.6 mg/mL. Additionally or alternatively, the concentration ofcitric acid in the reconstituted lyophilized daptomycin formulation isabout 57.6 mg/mL. The concentration of citric acid in the reconstitutedlyophilized daptomycin formulation can also be about 96.1 mg/mL.

In one embodiment, the additive is edetic acid. The concentration ofedetic acid in the reconstituted formulation is from about 1 mg/mL toabout 500 mg/mL, from about 1 mg/mL to about 250 mg/mL, or from about 1mg/mL to about 50 mg/mL.

In another embodiment, the additive is acetyl glucosamine. Theconcentration of acetyl glucosamine in the reconstituted lyophilizeddaptomycin formulation is about 52.5 mg/mL. Additionally oralternatively, the concentration of acetyl glucosamine in thereconstituted lyophilized daptomycin formulation is about 66.4 mg/mL.

In certain embodiments, upon reconstitution in a pharmaceuticallyacceptable diluent, the pH of the presently disclosed daptomycinformulations is in the range of from about 4.0 to about 7.0. In oneembodiment, the pH is in the range of from about 4.0 to about 5.0.

The purity of the daptomycin in the lyophilized daptomycin formulationsof the presently disclosed subject matter can be measured by any meansknown to one of ordinary skill in the art, including nuclear magneticresonance (NMR), or high performance liquid chromoatography coupled withUV (HPLC/UV) or mass spectrometry (HPLC/MS). In one embodiment, thepurity of the lyophilized daptomycin formulations is measured byreconstitution in a pharmaceutically acceptable diluent followed byanalysis employing HPLC/UV. The HPLC/UV process utilised for measuringthe purity of the lyophilized daptomycin formulations can be any methodknown to one of ordinary skill in the art, e.g., using appropriateHPLC/UV machines typically encountered in the industry, such as theHPLC/UV process described in US Patent 2007/191280 A1. In onenon-limiting example, the purity of daptomycin in a reconstitutedsolution of a lyophilized daptomycin formulation of the presentlydisclosed subject matter can be determined by HPLC/UV followed byanalysis of the peak area (the area-under-the curve-(AUC)) at awavelength of 223 nm for individual peaks of the chromatogram. Theamount of daptomycin can be measured with respect to the total amount ofimpurities present. Further, the relative amount of daptomycin withrespect to three of the known daptomycin degradants, specifically theanhydro-daptomycin, the β-isomer of daptomycin and the lactonehydrolysis product of daptomycin can be determined.

In one embodiment, the lyophilized daptomycin formulations of thepresently disclosed subject matter include daptomycin in a purity thatis higher than that of CUBICIN® product as determined by HPLC/UVanalysis of the reconstituted solution at a wavelength of 223 nm. Inanother embodiment, the lyophilized daptomycin formulations of thepresently disclosed subject matter include total impurities in an amountof less than that of the CUBICIN® product as determined by HPLC/UVanalysis of the reconstituted solution at a wavelength of 223 nm.

Methods of Using Lyophilized Daptomycin Formulations

The presently disclosed subject matter further provides a method oftreating a bacterial infection in a subject. The method includesadministering to a subject in need thereof, an effective amount of alyophilized daptomycin formulation including an additive selected fromthe group consisting of pharmaceutically acceptable antioxidants,pharmaceutically acceptable organic acids and pharmaceuticallyacceptable salts thereof, pharmaceutically acceptable glucosederivatives and a pharmaceutically acceptable salts thereof, andcombinations thereof.

The presently disclosed subject matter further provides a method oftreating or preventing a biofilm. The method includes exposing a surfaceof a device to a solution of an effective amount of a lyophilizeddaptomycin formulation including an additive selected from the groupconsisting of pharmaceutically acceptable antioxidants, pharmaceuticallyacceptable organic acids and pharmaceutically acceptable salts thereof,pharmaceutically acceptable glucose derivatives and a pharmaceuticallyacceptable salts thereof, and combinations thereof.

Methods of Preparing Lyophilized Daptomycin Formulations

Furthermore, the presently disclosed subject matter provides a methodfor preparing the presently disclosed lyophilized daptomycinformulation. The lyophilized daptomycin formulation can be lyophilizedfrom any solvent known to be suitable in the art. Acceptable solventsinclude, but are not limited to, water, aqueous butanol and aqueousethanol. The lyophilisation process utilised to prepare the presentlydisclosed lyophilized daptomycin formulations can be any method known toone of ordinary skill in the art using appropriate freeze dryingmachines typically encountered in the industry. Exemplary lyophilisationprocesses include those described in “Lyophilization: Introduction andBasic Principles” by Thomas A Jennings, InterPharm Press, 1999. In someembodiments, the method includes: (a) forming an aqueous solution of thedaptomycin and the additive selected from the group consisting ofpharmaceutically acceptable antioxidants, pharmaceutically acceptableorganic acids and pharmaceutically acceptable salts thereof,pharmaceutically acceptable glucose derivatives and a pharmaceuticallyacceptable salts thereof, and combinations thereof, (b) adjusting the pHto about 4.0 to about 5.0; and (c) lyophilising the solution to obtain alyophilisate. In one embodiment, the method includes: (a) forming anaqueous solution of the daptomycin at a pH of about 4.0 to about 5.0;(b) dissolving the additive selected from the group consisting ofpharmaceutically acceptable antioxidants, pharmaceutically acceptableorganic acids and pharmaceutically acceptable salts thereof,pharmaceutically acceptable glucose derivatives and a pharmaceuticallyacceptable salts thereof, and combinations thereof in the aqueoussolution of the daptomycin; (c) adjusting the pH to about 4.0 to about5.0; and (d) lyophilising the solution to obtain a powder. In anotherembodiment, the method includes: (a) forming an aqueous tertiary-butanolsolution of the daptomycin at a pH of 4.0 to 5.0; (b) dissolving anadditive selected from the group consisting of pharmaceuticallyacceptable antioxidants, pharmaceutically acceptable organic acids andpharmaceutically acceptable salts thereof, pharmaceutically acceptableglucose derivatives and a pharmaceutically acceptable salts thereof, andcombinations thereof in the aqueous/butanol solution of the daptomycin;(c) adjusting the pH to about 4.0 to about 5.0; and (d) lyophilising thesolution to obtain a powder.

EXAMPLES

The following examples are merely illustrative of the presentlydisclosed subject matter and they should not be considered as limitingthe scope of the invention in any way.

Example 1 I. Lyophilized Daptomycin Formulations

Table 1 provides examples of daptomycin formulation solutions that weresubsequently lyophilized to produce lyophilized formulations of thepresently disclosed subject matter including an additive selected fromthe group consisting of pharmaceutically acceptable antioxidants,pharmaceutically acceptable organic acids and pharmaceuticallyacceptable salts thereof, pharmaceutically acceptable glucosederivatives and pharmaceutically acceptable salts thereof, andcombinations thereof.

TABLE 1 Formulation Formulation Components A Daptomycin (350 mg/vial),Citric acid (56 mg/vial) NaOH (2M, 0.14 mL/vial) Water Additional NaOHto adjust pH B Daptomycin (350 mg/vial) Tartaric acid (84 mg/vial), NaOH(2M, 0.14 mL/vial) Water Additional NaOH to adjust pH C Daptomycin (350mg/vial), Succinic acid (67.2 mg/vial), NaOH (2M, 0.14 mL/vial) WaterAdditional NaOH to adjust pH D Daptomycin (350 mg/vial), Edetic acid(140 mg/vial), NaOH (2M, 0.14 mL/vial) Water Additional NaOH to adjustpH E Daptomycin (350 mg/vial), NaOH (2M, 0.14 mL/vial) ^(t)Butanol (1.12mL/vial) Water Additional NaOH to adjust pH F Daptomycin (350 mg/vial),Citric acid (56 mg/vial), Water

II. Analysis of Daptomycin Formulations

Reconstitution time was determined by injecting 7 mL of 0.9% sterilesodium chloride for injection to a vial including 350 mg of thelyophilized daptomycin formulation. The resultant mixture was swirledfor about 1 minute and allowed to stand. The reconstitution time is thetime required from addition of the diluent to total dissolution of thedaptomycin formulation.

Table 2 provides reconstitution times of the lyophilized daptomycinformulations including an additive in 0.9% sterile sodium chloride forinjection. Initial reconstitution times, and reconstitution after 1, 3or 6 months of storage at 5° C., 25° C. and 40° C. for the compositionsare provided. The pH of the reconstituted lyophilized daptomycinformulations is 4.7. The reconstitution times of the CUBICIN® productare included for comparison.

TABLE 2 Initial 3 months 6 months Formulation at 5° C. A 2 min 40 sec  1min 35 sec Not tested B 1 min 10 sec 2 min 0 sec Not tested C 3 min 20sec  5 min 50 sec Not tested D 2 min 30 sec  2 min 10 sec Not tested ENot tested Not tested Not tested CUBICIN ® 18 min 00 sec  21 min 30 sec13 min 00 sec Formulation at 25° C. A 2 min 40 sec  2 min 35 sec  2 min45 sec B 1 min 10 sec 1 min 0 sec Not tested C 3 min 20 sec  4 min 40sec Not tested D 2 min 30 sec 1 min 0 sec Not tested E 10 min 30 sec Not tested Not tested CUBICIN ® 18 min 00 sec  14 min 55 sec 14 min 42sec Formulation at 40° C. A 2 min 40 sec  1 min 50 sec Not tested B Nottested Not tested  0 min 55 sec C 1 min 10 sec 1 min 0 sec Not tested D3 min 20 sec 11 min 30 sec Not tested E 2 min 30 sec 1 min 0 sec Nottested F Not tested Not tested Not tested CUBICIN ® 18 min 00 sec  Nottested Not tested

The effect of pH on the reconstitution time of the daptomycinlyophilized powder including citric acid was assessed. Table 3 providesthe reconstitution times in 0.9% sterile sodium chloride for injectionof lyophilized daptomycin formulations including 350 mg daptomycin, 56mg citric acid and sodium hydroxide sufficient to adjust the pH of thelyophilisation solution upon reconstitution of the lyophilizedformulation. Initial reconstitution times and reconstitution times after3 months of storage at 40° C. for the compositions is provided.

TABLE 3 Formulation pH Initial 3 months 2.5 3 min 0 sec  2 min 30 sec3.0 3 min 0 sec  2 min 30 sec 3.3 >5 min 0 sec  3 min 15 sec 3.5 >5 min0 sec  4 min 30 sec 3.8 >5 min 0 sec  4 min 54 sec 4.0 1 min 50 sec 5min 00 sec 4.3 1 min 30 sec 2 min 20 sec 4.5 1 min 20 sec 3 min 28 sec4.8 2 min 20 sec 1 min 21 sec 5.0 2 min 0 sec  3 min 00 sec 5.3 3 min 0sec  2 min 20 sec 5.5 1 min 0 sec  1 min 20 sec 5.8 1 min 10 sec 1 min30 sec 6.0 1 min 10 sec 1 min 40 sec 6.3 1 min 0 sec  1 min 46 sec 6.5 0min 50 sec 1 min 30 sec 6.8 0 min 50 sec 1 min 10 sec 7.0 0 min 20 sec 0min 58 sec

The lyophilized daptomycin formulations of the presently disclosedsubject matter were tested for daptomycin stability as a relativemeasure of daptomycin degradant impurity levels. The lyophilizeddaptomycin formulations were reconstituted in 0.9% sterile sodiumchloride for injection the resultant solutions analysed by HPLC/UV. Theamount of daptomycin and impurities in the solution were determined by %peak area at a wavelength of 223 nm. The total amount of impurities wascalculated from the % peak area at a wavelength of 223 nm for all peaksother than that of daptomycin. The data are presented in the followingTables 4-9.

Table 4 displays the amount of total impurities, represented as % peakarea, for each composition at 1, 2, 3 and 6 months after storage at 5,25 and 40° C. The difference from the initial value is shown inparentheses.

TABLE 4 Total % Total % Total % Total % Total % Formulation ImpuritiesImpurities Impurities Impurities Impurities at 5° C. Initial 1 month 2months 3 months 6 months A 4.62 Not tested 4.82 (0.2)   4.42 (−0.20) Nottested B 4.64 Not tested Not tested 4.88 (0.24) Not tested C 4.97 Nottested Not tested  4.60 (−0.37) Not tested D 4.68 Not tested Not tested 4.52 (−0.16) Not tested CUBICIN ® 6.38 Not tested Not tested 6.57(0.19) 6.89 (0.51) Total Total Total Total Total Formulation ImpuritiesImpurities Impurities Impurities Impurities at 25° C. Initial 1 month 2months 3 months 6 months A 4.62 4.98 (0.36) 5.13 (0.51) 4.42 (−0.2) 5.28(0.66) B 4.64  4.62 (−0.02) 4.73 (0.09) 5.13 (0.49) Not tested C 4.97 4.59 (−0.38)  4.75 (−0.22) 5.13 (0.16) Not tested D 4.68  4.45 (−0.23) 4.60 (−0.08) 4.91 (0.23) Not tested CUBICIN ® 6.38 6.98 (0.60) 6.71(0.33) 7.18 (0.80) 7.87 (1.49) Total Total Total Total Total FormulationImpurities Impurities Impurities Impurities Impurities at 40° C. Initial1 month 2 months 3 months 6 months A 4.62 4.68 (0.06) Not tested 5.83(1.21) Not tested B 4.64 5.29 (0.65) 5.36 (0.72) 5.77 (1.13) Not testedC 4.97 5.98 (1.01) 6.91 (1.94) 7.79 (2.82) Not tested D 4.68 4.97 (0.29)5.38 (0.70) 5.89 (1.21) Not tested CUBICIN ® 6.38 Not tested Not testedNot tested Not tested

Table 5 displays the amount of the anhdyro daptomycin impurities,represented as % peak area, for each composition at 1, 2, 3 and 6 monthsafter storage at 5, 25 and 40° C. The difference from the initial valueis shown in parentheses.

TABLE 5 Anhydro Anhydro Anhydro Anhydro Anhydro Impurity ImpurityImpurity Impurity Impurity Initial 1 month 2 months 3 months 6 monthsFormulation at 5° C. A 1.18 Not tested  1.1 (−0.08)  1.06 (−0.12) Nottested B 0.97 Not tested Not tested 1.00 (0.03) Not tested C 1.08 Nottested Not tested  1.04 (−0.04) Not tested D 1.11 Not tested Not tested 1.09 (−0.02) Not tested CUBICIN ® 1.71 Not tested Not tested  1.69(−0.02) 2.06 (0.35) Formulation at 25° C. A 1.18 1.19 (0.01) 1.29 (0.11)1.22 (0.04) 1.45 (0.27) B 0.97 1.04 (0.07) 1.11 (0.14) 1.18 (0.21) Nottested C 1.08 1.16 (0.08) 1.27 (0.19) 1.39 (0.31) Not tested D 1.11 1.15(0.04) 1.16 (0.05) 1.32 (0.21) Not tested CUBICIN ® 1.71 1.92 (0.21)2.23 (0.52) 2.06 (0.35) 2.56 (0.85) Formulation at 40° C. A 1.18 1.48(0.30) Not tested 1.72 (0.54) Not tested B 0.97 1.33 (0.36) 1.49 (0.52)1.63 (0.66) Not tested C 1.08 1.71 (0.63) 2.05 (0.97)  2.49 (2.41)) Nottested D 1.11 1.50 (0.39) 1.71 (0.60) 1.87 (0.76) Not tested CUBICIN ®1.71 Not tested Not tested Not tested Not tested

Table 6 displays the amount of the hydrolysis daptomycin impurities,represented as % peak area, for each composition at 1, 2, 3 and 6 monthsafter storage at 5, 25 and 40° C. The difference from the initial valueis shown in parentheses.

TABLE 6 Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis ImpurityImpurity Impurity Impurity Impurity Initial 1 month 2 months 3 months 6months Formulation at 5° C. A 0.52 Not tested 0.57 (0.05) 0.53 (0.01)Not tested B 0.73 Not tested Not tested  0.68 (−0.05) Not tested C 0.62Not tested Not tested  0.53 (−0.09) Not tested D 0.51 Not tested Nottested  0.43 (−0.08) Not tested CUBICIN ® 0.40 Not tested Not tested0.44 (0.04) 0.45 (0.05) Formulation at 25° C. A 0.52 0.61 (0.09) 0.64(0.12) 0.61 (0.09) 0.62 (0.10) B 0.73 0.75 (0.02) 0.79 (0.06) 0.76(0.03) Not tested C 0.62 0.66 (0.04) 0.71 (0.09) 0.77 (0.15) Not testedD 0.51 0.52 (0.01) 0.55 (0.04) 0.53 (0.02) Not tested CUBICIN ® 0.400.49 (0.09) 0.51 (0.11) 0.56 (0.16) 0.65 (0.25) Formulation at 40° C. A0.52 0.66 (0.14) Not tested 0.86 (0.34) Not tested B 0.73 0.89 (0.16)0.96 (0.23) 1.02 (0.29) Not tested C 0.62 0.98 (0.36) 1.18 (0.56) 1.39(0.77) Not tested D 0.51 0.64 (0.13) 0.73 (0.22) 0.80 (0.29) Not testedCUBICIN ® 0.40 Not tested Not tested Not tested Not tested

Table 7 displays the amount of the β-isomer daptomycin impurities,represented as % peak area, for each composition at 1, 2, 3 and 6 monthsafter storage at 5, 25 and 40° C. The difference from the initial valueis shown in parentheses.

TABLE 7 β-Isomer β-Isomer β-Isomer β-Isomer β-Isomer Impurity ImpurityImpurity Impurity Impurity Initial 1 month 2 months 3 months 6 monthsFormulation at 5° C. A 0.56 Not tested  0.6 (0.04) 0.62 (0.06) Nottested B 0.64 Not tested Not tested 0.70 (0.06) Not tested C 0.62 Nottested Not tested 0.63 (0.01) Not tested D 0.57 Not tested Not tested0.60 (0.03) Not tested CUBICIN ® 1.15 Not tested Not tested 1.17 (0.02)1.17 (0.02) Formulation at 25° C. A 0.56  0.6 (0.04)  0.6 (0.04) 0.61(0.05) 0.64 (0.08) B 0.64 0.67 (0.03) 0.67 (0.03) 0.69 (0.05) Not testedC 0.62 0.62 (0)   0.62 (0.00) 0.63 (0.01) Not tested D 0.57 0.59 (0.02)0.59 (0.02) 0.60 (0.03) Not tested CUBICIN ® 1.15 1.16 (0.01) 1.20(0.05) 1.17 (0.02) 1.19 (0.04) Formulation at 40° C. A 0.56  0.54(−0.02) Not tested 0.63 (0.07) Not tested B 0.53 Not tested Not testedNot tested 0.59 (0.06) C 0.64 0.69 (0.05) 0.68 (0.04) 0.69 (0.05) Nottested D 0.62 0.64 (0.02) 0.64 (0.02) 0.65 (0.03) Not tested E 0.57 0.60(0.03) 0.61 (0.04) 0.62 (0.05) Not tested CUBICIN ® 1.15 Not tested Nottested Not tested Not tested

The effect of pH on the stability of the lyophilized daptomycinformulations including citric acid were tested as a relative measure ofdaptomycin degradant impurity levels. The lyophilized daptomycinformulations, including 350 mg daptomycin, 56 mg citric acid, and sodiumhydroxide sufficient to adjust the pH of the lyophilisation solution,were reconstituted in 0.9% sterile sodium chloride for injection and theresultant solutions analysed by HPLC/UV. Table 8 displays the amount ofdaptomycin impurities, represented as % peak area, for each compositionupon initial preparation and after storage at 25° C. for 3 months. Thedifference from the initial value is shown in parentheses.

TABLE 8 Anhydro Anhydro Hydrolysis Hydrolysis β-Isomer β-IsomerImpurities Impurities Impurities Impurities Impurities Impurities pHInitial 3 months Initial 3 months Initial 3 months 2.5 0.83 1.10 (0.27)0.40  0.37 (−0.03) 0.57 0.51 (−0.06) 3.0 0.88 1.17 (0.29) 0.45  0.42(−0.03) 0.58 0.52 (−0.06) 3.3 0.88 1.19 (0.31) 0.46  0.43 (−0.03) 0.580.53 (−0.05) 3.5 0.90 1.27 (0.37) 0.47  0.46 (−0.01) 0.58 0.54 (−0.04)3.8 0.93 1.32 (0.39) 0.49 0.49 (0.00) 0.59 0.54 (−0.05) 4.0 0.93 1.25(0.32) 0.53 0.53 (0.00) 0.60 0.56 (−0.04) 4.3 0.94 1.26 (0.32) 0.55 0.58(0.03) 0.61 0.58 (−0.03) 4.5 0.96 1.27 (0.31) 0.58 0.62 (0.04) 0.62 0.58(−0.04) 4.8 0.95 1.26 (0.31) 0.64 0.72 (0.08) 0.65 0.61 (−0.04) 5.0 0.951.23 (0.28) 0.64 0.76 (0.12) 0.65 0.62 (−0.03) 5.3 0.90 1.15 (0.25) 0.690.85 (0.16) 0.66 0.63 (−0.03) 5.5 0.87 1.10 (0.23) 0.74 0.95 (0.21) 0.680.65 (−0.03) 5.8 0.83 1.01 (0.18) 0.84 1.18 (0.34) 0.72 0.67 (−0.05) 6.00.80 0.94 (0.14) 1.01 1.42 (0.41) 0.74 0.69 (−0.05) 6.3 0.74 0.89 (0.15)1.09 1.59 (0.50) 0.76 0.72 (−0.04) 6.5 0.69 0.86 (0.17) 1.16 1.80 (0.64)0.77 0.76 (−0.01) 6.8 0.65 0.80 (0.15) 1.20 1.90 (0.70) 0.77 0.74(−0.03) 7.0 0.64 0.78 (0.14) 1.24 2.00 (0.76) 0.79 0.83 (0.04) 

Table 9 displays the total amount of all daptomycin impurities,represented as % peak area, for each composition upon initialpreparation and after storage at 25° C. for 3 months. The differencefrom the initial value is shown in parentheses.

TABLE 9 Total Total Impurities Impurities pH Initial 3 months 2.5 4.026.12 (2.10) 3.0 4.18 5.68 (1.50) 3.3 4.15 5.32 (1.17) 3.5 4.21 5.37(1.16) 3.8 4.13 5.24 (1.11) 4.0 4.32 5.16 (0.84) 4.3 4.20 5.19 (0.99)4.5 4.31 5.21 (0.90) 4.8 4.54 5.29 (0.75) 5.0 4.36 5.34 (0.98) 5.3 4.395.34 (0.95) 5.5 4.57 5.45 (0.88) 5.8 4.78 5.40 (0.62) 6.0 4.74 5.57(0.83) 6.3 4.79 5.80 (1.01) 6.5 4.97 6.60 (1.63) 6.8 4.77 5.90 (1.13)7.0 4.84 6.19 (1.35)

Example 2

The osmolalites of the presently disclosed lyophilized daptomycinformulations including citric acid (175 mM, 237.5 mM, 300 mM or 500 mM),ascorbic acid (237.5 mM or 300 mM) and 62.5 mg/mL daptomycin wereassessed. Table 10 provides the osmolalities of the lyophilizeddaptomycin formulations including ascorbic acid, citric acid in eithersterile water for injection (“WFI”) or a 0.9% sodium chloride solutionfor injection. The pH of the reconstituted lyophilized daptomycinformulations was in the range of 4.45 to 4.74. The osmolalities of theCUBICIN® product and of a benchmark sucrose daptomycin formulation wereincluded for comparison.

TABLE 10 Osmolality (mOsmol/kg) Sucrose Citric acid Ascorbic acidInfusion solution CUBICIN ® 438 175 237.5 300 500 237.5 300concentration CDF049M mM mM mM mM mM mM mM NaCl IV Bolus 338 712 642 740828 1156 636 705 reconstitution (50 mg/mL) IV Infusion 310 444 435 473510 628 425 453 (20 mg/mL) IV Infusion 302  36 364 383 400 464 357 367(10 mg/mL) WFI IV Bolus 49 427 Not 458 572 940 343 421 reconstitution(50 mg/mL) tested IV Infusion 198 Not Not Not 398 562 315 344 (20 mg/mL)tested tested tested IV Infusion 249 313 Not 327 344 422 306 321 (10mg/mL) tested

The osmolalites of the ascorbic acid daptomycin formulations were lowerthan those of the citric acid daptomycin formulations.

Example 3

The reconstitution times, osmolalities, color appearances andstabilities of the presently disclosed lyophilized daptomycinformulations, e.g., including ascorbic acid, citric acid, and acetylglucosamine as an additive and 62.5 mg/mL daptomycin, were assessed.

Reconstitution time was determined by injecting 7 mL of a 0.9% sodiumchloride solution to a vial containing 350 mg/vial of lyophilizeddaptomycin. The resultant mixture was swirled for one minute and allowedto stand. The reconstitution time is the time required for addition ofthe diluent to total dissolution of the lyophilized daptomycinformulation.

TABLE 11 Average recon- Average recon- Average recon- stitution time forstitution time for stitution time for a lyophilized a lyophilized alyophilized daptomycin formula- daptomycin formula- daptomycin formula-Concentration tion including tion including tion including of theadditive citric acid ascorbic acid acetyl glucosamine Sucrose CUBICIN ®237.5 mM 1 m 55 s 1 m 45 s 3 m 39 s 1 m 01 s 23 m 44 s (all samples,(all samples) (all samples, (all samples) (all samples) n = 15)¹ (n =15) n = 15)² (n = 8) (n = 10)  300 mM 2 m 14 s 1 m 57 s 3 m 8 s (allsamples, (all samples, (all samples, n = 30)¹ n = 30)³ n = 30)⁴ ¹2samples had a reconstitution time of >5 min ²3 samples had areconstitution time of >5 min ³1 sample had a reconstitution time of >5min ⁴6 samples had a reconstitution time of >5 min

Table 11 provides the average reconstitution times of the lyophilizeddaptomycin formulations including ascorbic acid, citric acid, and acetylglucosamine in a 0.9% sodium chloride solution for injection. The pH ofthe reconstituted lyophilized daptomycin formulations was in the rangeof 4.45 to 4.74. The reconstitution times of the CUBICIN® product and ofa benchmark sucrose daptomycin formulation were included for comparison.

Table 12 provides the osmolalities of the lyophilized daptomycinformulations including ascorbic acid, citric acid and acetylglucosamine. These formulations were reconstituted with a 0.9% sterilesodium chloride solution for injection and WFI. The osmolalities of theCUBICIN® product and of a benchmark sucrose daptomycin formulation wereincluded for comparison, as shown in Table 13.

TABLE 12 Osmolality of Osmolality of Osmolality of a lyophilized alyophilized a lyophilized daptomycin formula- daptomycin formula-daptomycin formula- Concentration tion including tion including tionincluding of the additive Administration citric acid ascorbic acidacetyl glucosamine 0.9% Sodium Chloride reconstitution (mOsmol/Kg) 237.5mM Bolus (50 mg/mL) 740 636 548 Infusion (10 mg/mL) 383 357 339 WFIreconstitution (mOsmol/Kg) Bolus (50 mg/mL) 458 343 249 Infusion (10mg/mL) 327 306 289 0.9% Sodium Chloride reconstitution (mOsmol/Kg)  300mM Bolus (50 mg/mL) 828 705 602 Infusion (10 mg/mL) 400 367 346 WFIreconstitution (mOsmol/Kg) Bolus (50 mg/mL) 572 421 305 Infusion (10mg/mL) 344 321 297

TABLE 13 Osmolality of Osmolality of Administration Sucrose CUBICIN ®0.9% Sodium Chloride reconstitution (mOsmol/Kg) Bolus (50 mg/mL) 712 338Infusion (10 mg/mL) 366 302 WFI reconstitution (mOsmol/Kg) Bolus (50mg/mL) 427 49 Infusion (10 mg/mL) 313 249

Table 14 provides the initial color appearances of the lyophilizeddaptomycin formulations including ascorbic acid, citric acid and acetylglucosamine at a 50 mg/mL bolus or 10 mg/mL infusion solution exposed toa temperature of ambient laboratory temperature (about 20° C.) and alighting condition of ambient laboratory lighting (about 400 lux). Thecolor appearances of the CUBICIN® product and a benchmark sucrosedaptomycin formulation were included for comparison.

TABLE 14 Acetyl Additive Citric Ascorbic Glucos- concentration acid acidamine Sucrose CUBICIN ® 237.5 Mm Light Light Light Light Light  300 mMyellow yellow to yellow yellow yellow light brown

The lyophilized daptomycin formulations including ascorbic acid (237.5mM and 300 mM), citric acid (237.5 mM and 300 mM), or acetyl glucosamine(237.5 mM and 300 mM), and 62.5 mg/mL daptomycin were tested fordaptomycin stability as a relative measure of daptomycin degradantimpurity level. The amount of daptomycin and impurities in the solutionwere determined by the % peak area at a wavelength of 223 nm. Table 15displays the amount of daptomycin impurities, represented as % peakarea, for each composition upon initial preparation and after storage at25° C. and 40° C. for 2, 3 and 6 months. The difference from the initialvalue was shown in parentheses. The impurities of the CUBICIN® productand a benchmark sucrose daptomycin formulation were included forcomparison.

TABLE 15 Impurity % Temperature Additive Time point Anhydro- (° C.)Concentration Additive (month) Hydrolysis Daptomycin Impurity 1 B isomer25° C. 237.5 mM  Citric Acid T = 0 0.43 1.05 0.12 0.57 T = 2 0.48 (0.06) 1.02 (−0.03) 0.07 (−0.05) 0.57 (0.0)  T = 3 0.46 (0.03) 1.10 (0.05)0.07 (−0.05) 0.52 (−0.05) T = 6 0.43 (0.00) 1.11 (0.06) 0.07 (−0.06)0.53 (−0.04) Ascorbic Acid T = 0 0.43 1.11 0.12 0.60 T = 2 0.52 (0.10)1.14 (0.03) 0.14 (0.02)  0.53 (−0.07) T = 3 0.52 (0.09) 1.12 (0.01) 0.12(0.00)  0.53 (−0.07) T = 6 0.46 (0.04) 1.14 (0.03) 0.29 (0.17)  0.55(−0.05) Acetyl T = 0 0.41 0.97 0.10 0.56 Glucosamine T = 2 0.45 (0.04)1.04 (0.07) 0.07 (−0.03) 0.52 (−0.04) T = 3 0.46 (0.05) 1.10 (0.13) 0.07(−0.03) 0.52 (−0.04) T = 6 0.43 (0.03) 1.07 (0.10) 0.12 (0.02)  0.60(0.04)  300 mM Citric Acid T = 0 0.40 1.05 0.12 0.56 T = 2 0.46 (0.06)1.03 (0.02) 0.08 (−0.04) 0.56 (0.00)  T = 3 0.44 (0.04) 1.13 (0.08) 0.07(−0.04) 0.51 (−0.05) T = 6 0.40 (0.0)  1.06 (0.01) 0.06 (−0.05) 0.51(−0.05) Ascorbic Acid T = 0 0.42 1.13 0.11 0.59 T = 2 0.50 (0.08) 1.16(0.03) 0.12 (0.02)  0.53 (−0.06) T = 3 0.49 (0.07)  1.12 (−0.01) 0.11(0.0)  0.53 (−0.06) T = 6 0.45 (0.03)  1.12 (−0.01) 0.22 (0.12)  0.54(−0.05) Acetyl T = 0 0.40 0.97 0.10 0.59 Glucosamine T = 2 0.44 (0.04)1.03 (0.05) 0.07 (−0.03) 0.52 (−0.07) T = 3 0.44 (0.04) 1.13 (0.16) 0.07(−0.03) 0.51 (−0.07) T = 6 0.42 (0.02) 1.05 (0.08) 0.12 (0.02)  0.59(0.0)  438 mM Sucrose T = 0 0.53 0.91 0.08 0.53 (Benchmark T = 2 0.67(0.14) 0.99 (0.08) 0.08 (0.0)  0.59 (0.06)  formulation) T = 3 0.58(0.05) 0.95 (0.04) 0.06 (−0.02) 0.52 (−0.01) T = 6 0.55 (0.02) 0.98(0.07) 0.12 (0.04)  0.48 (−0.05) Cubicin ® T = 0 0.79 2.38 0.28 1.13 T =2  0.58 (−0.21)  1.87 (−0.51) 0.40 (0.12)  1.15 (0.02)  T = 3  0.60(−0.19)  1.94 (−0.44) 0.19 (−0.09) 1.08 (−0.05) T = 6  0.67 (−0.12) 2.17 (−0.21) 0.26 (−0.02) 1.16 (0.03)  40° C. 237.5 mM  Citric Acid T =0 0.43 1.05 0.12 0.57 T = 1 0.48 (0.05) 1.17 (0.12) 0.08 (−0.05) 0.55(−0.03) T = 2 0.51 (0.09) 1.26 (0.21) 0.06 (−0.06) 0.59 (0.01)  T = 30.52 (0.09) 1.27 (0.22) 0.04 (−0.08) 0.51 (−0.06) T = 6 0.57 (0.14) 1.44(0.39) 0.12 (0.0)  0.54 (−0.03) Ascorbic Acid T = 0 0.43 1.11 0.12 0.60T = 1 0.59 (0.16) 1.25 (0.14) 0.19 (0.07)  0.61 (0.01)  T = 2 0.63(0.21) 1.30 (0.20) 0.31 (0.19)  0.63 (−0.03) T = 3 0.66 (0.23) 1.32(0.22) 0.22 (0.10)  0.57 (−0.03) T = 6 0.66 (0.23) 1.40 (0.29) 0.27(0.15)  0.71 (0.11)  Acetyl T = 0 0.41 0.97 0.10 0.56 Glucosamine T = 10.47 (0.06) 1.13 (0.16) 0.08 (−0.02) 0.54 (−0.02) T = 2 0.49 (0.09) 1.20(0.23) 0.09 (−0.01) 0.64 (0.08)  T = 3 0.50 (0.09) 1.26 (0.29) 0.04(−0.06) 0.56 (0.0)  T = 6 0.54 (0.13) 1.52 (0.55) 0.26 (0.16)  0.71(0.15)  300 mM Citric Acid T = 0 0.40 1.05 0.12 0.56 T = 1 0.44 (0.04)1.19 (0.13) 0.08 (−0.05) 0.54 (−0.02) T = 2 0.47 (0.07) 1.28 (0.23) 0.06(−0.06) 0.55 (−0.02) T = 3 0.48 (0.08) 1.25 (0.20) 0.05 (−0.07) 0.50(−0.06) T = 6 0.50 (0.10) 1.40 (0.35) 0.14 (0.02)  0.55 (−0.01) AscorbicAcid T = 0 0.42 1.13 0.11 0.59 T = 1 0.55 (0.13) 1.24 (0.11) 0.16(0.06)  0.60 (0.01)  T = 2 0.60 (0.18) 1.28 (0.15) 0.25 (0.14)  0.62(0.03)  T = 3 0.59 (0.17) 1.29 (0.16) 0.18 (0.07)  0.56 (−0.03) T = 60.60 (0.18) 1.32 (0.19) 0.26 (0.15)  0.69 (0.10)  Acetyl T = 0 0.40 0.970.10 0.59 Glucosamine T = 1 0.46 (0.06) 1.11 (0.14) 0.08 (−0.02) 0.54(−0.05) T = 2 0.48 (0.08) 1.16 (0.19) 0.09 (−0.01) 0.60 (0.01)  T = 30.47 (0.07) 1.19 (0.22) 0.09 (−0.01) 0.55 (−0.04) T = 6 0.57 (0.17) 1.40(0.43) 0.11 (0.01)  0.65 (0.06)  438 mM Sucrose T = 0 0.53 0.91 0.080.53 (Benchmark T = 1 0.59 (0.06) 1.05 (0.14) 0.06 (−0.02) 0.64 (0.31) formulation) T = 2 0.67 (0.14) 1.13 (0.22) 0.06 (−0.02) 0.58 (0.05)  T =3 0.59 (0.06) 1.11 (0.20) 0.15 (0.07)  0.53 (0.0)  T = 6 0.55 (0.02)1.24 (0.33) 0.14 (0.06)  0.48 (−0.05) Cubicin ® T = 0 0.51 1.55 0.301.14 T = 1 0.79 (0.28) 2.38 (0.83) 0.28 (−0.02) 1.13 (−0.01) T = 2 0.94(0.43) 2.80 (1.25) 0.29 (−0.02) 1.16 (0.02)  T = 3 1.04 (0.53) 3.20(1.65) 0.22 (0.07)  1.10 (−0.04) T = 6 1.24 (0.73) 3.70 (2.15) N/A 1.20(0.06) 

As shown in Table 15, when stored at 25° C. for 6 months, the anhydrodaptomycin impurities increased up to about 0.1% for all formulationscompared to the initial value, which was comparable to the benchmarkSucrose daptomycin formulation. Impurity 1 increased by about 0.2% forboth 237.5 mM and 300 mM lyophilised ascorbic acid daptomycinformulations, by about 0.02% for both 237.5 mM and 300 mM lyophilisedacetyl glucosamine daptomycin formulations, while remained stable forboth 237.5 mM and 300 mM lyophilised citric acid daptomycinformulations, which was comparable to benchmark Sucrose daptomycinformulation and the CUBICIN® product. All other major impuritiesremained stable without significant increases. The β-isomer daptomycinimpurity remained stable for almost all formulations.

When stored at 40° C. for 6 months, differences in the increases ofanhydro daptomycin impurities were observed between citric acidformulations and ascorbic acid formulations. Impurity 1 & β-isomerdaptomycin impurities remained stable for the citric acid formulations,however increased up to 0.2% & 0.1%, respectively, for the ascorbic acidformulations, compared to the initial value. Hydrolysis impurityincreased up to 0.14% for the citric acid formulations (0.14% for the237.5 mM formulation, and 0.1% for the 300 mM formulation). Hydrolysisimpurity increased up to 0.23% for the ascorbic acid formulations (0.23%for the 237.5 mM formulation, and 0.18% for the 300 mM formulation).Hydrolysis impurity increased up to 0.17% for the acetyl glucosamineformulations (0.14% for the 237.5 mM formulation, and 0.17% for the 300mM formulation). Anhydro daptomycin impurities increased up to 0.39% forthe citric acid formulations and up to 0.29% for the ascorbic acidformulations, which was comparable to the Sucrose formulation. The rateof increase for anhydro daptomycin impurities was the lowest for the 300mM ascorbic acid formulation. The rates of increase for all other majorimpurities were approximately double for ascorbic acid formulationscompared to citric acid formulations.

The above-presented stability data showed that citric acid lyophilizeddaptomycin formulations have a slight advantage over ascorbic acidlyophilized daptomycin formulations and acetyl glucosamine lyophilizeddaptomycin formulations as citric acid lyophilized daptomycinformulations would tolerate heat excursions better than ascorbic acidlyophilized daptomycin formulations and acetyl glucosamine lyophilizeddaptomycin formulations.

The impurity amounts or stability for the 237.5 mM formulations werecomparable to the 300 mM formulations, e.g., the 237.5 mM formulationwas not significantly different from the 300 mM formulation for citricacid. In some examples, a 237.5 mM formulation concentration ispreferred at least because it has lower osmolality, and it is possiblyeasy to register lower the amounts of the additives.

Example 4

The color appearances of the presently disclosed lyophilized daptomycinformulations, e.g., including ascorbic acid (237.5 mM and 300 mM) orcitric acid (237.5 mM and 300 mM) and 62.5 mg/mL daptomycin at 50 mg/mLIV bolus or 10 mg/mL IV infusion at initial, 4, 24, and 48 hours exposedto room temperature and lighting conditions of about 400 lux, wereassessed. The color appearances for the CUBICIN® product and for abenchmark sucrose daptomycin formulation were included for comparison.The lyophilized daptomycin formulations and the benchmark sucrosedaptomycin formulation were reconstituted in both 0.9% NaCl and WFI.

In a 50 mg/mL IV bolus solution, the general color trend of alldaptomycin formulations was a light yellow at initial and four hoursfollowed by an increase in color intensity to yellow at 24 to 48 hours.The benchmark sucrose daptomycin formulation was a slightly dull/lessintense yellow. Both 237.5 mM and 300 mM citric acid daptomycinformulations were equivalent to the CUBICIN® product in color. Both237.5 mM and 300 mM ascorbic acid daptomycin formulations were slightlymore intense yellow in color than the CUBICIN® product at 24 to 48hours.

10 mg/mL IV infusion solution, the general color trend of all solutionswas light yellow, which remained light yellow over time to 48 hours. Thebenchmark sucrose daptomycin formulation was slightly less intenseyellow. Both 237.5 mM and 300 mM citric acid daptomycin formulationswere equivalent to the CUBICIN® product in color. Both 237.5 mM and 300mM ascorbic acid daptomycin formulations were slightly lighter then theCUBICIN® product at 24 to 48 hours.

Therefore, all reconstituted solutions were essentially yellow with anyvariation considered different shades of yellow compared to the yellowof the CUBICIN® product. The color differences between the ascorbic aciddaptomycin formulations and the CUBICIN® product were subtle. Therefore,the reconstituted solution color of both citric acid and ascorbic aciddaptomycin formulations are acceptable.

The presently disclosed subject matter is not to be limited in scope bythe specific embodiments described herein. Indeed, various modificationsof the presently disclosed subject matter in addition to those describedherein will become apparent to those skilled in the art from theforegoing description. Such modifications are intended to fall withinthe scope of the appended claims.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of the application.

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes to the sameextent as if each was so individually denoted.

What is claimed is:
 1. A lyophilized daptomycin formulation comprising alyophilizate of an aqueous solution of daptomycin and an additiveselected from the group consisting of pharmaceutically acceptableorganic acids and pharmaceutically acceptable salts thereof, acetylglucosamine, and combinations thereof, wherein the pharmaceuticallyacceptable organic acids and pharmaceutically acceptable salts thereof,if present, have a concentration of from 10 mM to 500 mM in the aqueoussolution prior to lyophilization.
 2. The lyophilized daptomycinformulation of claim 1, wherein the formulation comprises from about 200mg to about 600 mg of daptomycin.
 3. The lyophilized daptomycinformulation of claim 1, wherein the pharmaceutically acceptable organicacid is selected from the group consisting of monocarboxylic organicacids, dicarboxylic organic acids, hydroxyl substituted dicarboxylicorganic acids, tricarboxylic organic acids, hydroxyl substitutedtricarboxylic organic acids, tetracarboxylic organic acids, andcombinations thereof.
 4. The lyophilized daptomycin formulation of claim3, wherein the pharmaceutically acceptable organic acid is a hydroxylsubstituted tricarboxylic organic acid.
 5. The lyophilized daptomycinformulation of claim 4, wherein the hydroxyl substituted tricarboxylicorganic acid is citric acid.
 6. A reconstituted daptomycin formulationcomprising the lyophilized daptomycin formulation of claim 1, whereinthe lyophilized formulation is reconstituted in a pharmaceuticallyacceptable diluent to obtain the reconstituted daptomycin formulation.7. The reconstituted daptomycin formulation of claim 6, wherein thelyophilized formulation is reconstituted in the pharmaceuticallyacceptable diluent in less than about 5 minutes.
 8. The reconstituteddaptomycin formulation of claim 6, wherein the concentration ofdaptomycin in the reconstituted daptomycin formulation is from about 20mg/mL to about 100 mg/mL.
 9. The reconstituted daptomycin formulation ofclaim 6, wherein the concentration of the additive in the reconstituteddaptomycin formulation is from about 1 mM to about 500 mM.
 10. Thereconstituted daptomycin formulation of claim 9, wherein theconcentration of the additive in the reconstituted daptomycinformulation is about 237.5 mM.
 11. The reconstituted daptomycinformulation of claim 9, wherein the concentration of the additive in thereconstituted daptomycin formulation is about 300 mM.
 12. Thereconstituted daptomycin formulation of claim 6, wherein theconcentration of the additive in the reconstituted daptomycinformulation is from about 1 mg/mL to about 500 mg/mL.
 13. Thereconstituted daptomycin formulation of claim 6, wherein the pH of thereconstituted daptomycin formulation is from about 4.0 to about 5.0. 14.The reconstituted daptomycin formulation of claim 13, wherein the pH ofthe reconstituted daptomycin formulation is about 4.7.
 15. Thereconstituted daptomycin formulation of claim 6, wherein the diluent isselected from the group consisting of sterile water for injection,bacteriostatic water for injection, 0.45% sodium chloride solution forinjection, 0.9% sodium chloride solution for injection, Ringer'ssolution, lactated Ringer's solution, and combinations thereof.
 16. Thereconstituted daptomycin formulation of claim 15, wherein the diluent is0.9% sterile sodium chloride solution for injection.
 17. Thereconstituted daptomycin formulation of claim 15, wherein the diluent issterile water for injection.
 18. A method of treating a bacterialinfection in a subject, comprising administering to the subject aneffective amount of a reconstituted daptomycin formulation of claim 6.19. A method of treating a biofilm, the method comprising exposing asurface of a device to a solution comprising an effective amount of areconstituted daptomycin formulation of claim
 6. 20. A method forpreparing a lyophilized daptomycin formulation of claim 1, wherein themethod comprises: (a) forming an aqueous solution of daptomycin and anadditive, which is selected from the group consisting ofpharmaceutically acceptable organic acids and pharmaceuticallyacceptable salts thereof, acetyl glucosamine, and combinations thereof,wherein the pharmaceutically acceptable organic acids andpharmaceutically acceptable salts thereof, if present, have aconcentration of from 10 mM to 500 mM in the aqueous solution; (b)adjusting the pH to from about 4.0 to about 5.0; and (c) lyophilizingthe solution to obtain a lyophilizate.
 21. A method for preparing alyophilized daptomycin formulation of claim 1, wherein the methodcomprises: (a) forming an aqueous solution of daptomycin at a pH of fromabout 4.0 to 5.0; (b) dissolving an additive selected from the groupconsisting of pharmaceutically acceptable organic acids,pharmaceutically acceptable salts thereof and acetyl glucosamine in theaqueous solution of the daptomycin, wherein the pharmaceuticallyacceptable organic acids and pharmaceutically acceptable salts thereof,if present, have a concentration of from 10 mM to 500 mM in the aqueoussolution; (c) adjusting the pH to from about 4.0 to about 5.0; and (d)lyophilising lyophilizing the solution to obtain a powder.